Batch Purification of Proteins

A crude protein extract derived from some type of physical or chemical manipulation of a source will typically contain several types of contaminating biomolecules. These contaminants can include carbohydrates, lipids, nucleic acids, proteins, salts, and other cellular debris. The separation of the protein fraction of the extract from these contaminants is usually referred to as the capture step and is typically performed early in the purification process. The desired protein is then separated from the other captured proteins in subsequent steps using higher resolution purification techniques.Two popular methods of protein capture are bulk precipitation and batch chromatography. Bulk precipitation methods typically involve salting out the protein fraction by the addition of chaotropic salts, such as ammonium sulfate. The protein fraction can also be precipitated by the addition of certain organic solvents or by long chain synthetic polymers. Batch chromatography involves the binding of the protein fraction to some type of chromatographic gel, followed by filtration to remove contaminants, and, finally, elution and collection of the captured proteins.The crude α-galactosidase extract has now been prepared, and the conditions for maintaining activity have been investigated. α-Galactosidase will now be isolated, or captured, from the crude extract prior to its purification and analysis. This capture will effectively separate gross contaminants such as cellular debris, nucleic acids, and lipids from α-galactosidase. The experimental section in this chapter will consist of the capture of the α-galactosidase component from the crude extract by batch ion exchange chromatography.